Aug 09, 2023

Public workspaceReverse-phase high pH fractionation (using Thermo Fisher, Cat# 84868)

DOI
dx.doi.org/10.17504/protocols.io.yxmvm32bnl3p/v1
  • 1Lazarou Lab, WEHI
Louise Uoselis
Open access
DOI: dx.doi.org/10.17504/protocols.io.yxmvm32bnl3p/v1
Protocol Citation: Louise Uoselis 2023. Reverse-phase high pH fractionation (using Thermo Fisher, Cat# 84868). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm32bnl3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86212
Keywords: ASAPCRN
Abstract
This protocol uses the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher, Cat# 84868)
Conditioning the columns
Conditioning the columns
4m
Remove the white cap on the end of the column and place the column in a 2 mL collection tube.
Centrifuge at Centrifigation5000 rcf, 00:02:00 at TemperatureRoom temperature , discard the liquid.

2m
Remove the screw cap and add Amount300 µL acetonitrile (ACN) to the column (replacing the screw cap after).

Centrifuge at Centrifigation5000 rcf, 00:02:00 at TemperatureRoom temperature , discard the liquid.

2m
Repeat steps 3 and 4 (for a total of 2 washes with ACN).
Repeat steps 3 – 5 with 0.1% TFA instead of ACN (total of 2x washes with 0.1% TFA).
The column is now ready to use.
Fractionating the samples
Fractionating the samples
16m 20s
Add Amount300 µL of 0.1% v/v trifluoroacetic acid to each sample.

Vortex for ~ Duration00:00:10

10s
Leave to incubate for Duration00:05:00 at TemperatureRoom temperature

5m
Vortex for ~ Duration00:00:10

10s
Sonicate in a waterbath sonicator for Duration00:05:00 in an ice slurry.

5m
Load each sample into a fractionation column, replace the cap and centrifuge at Centrifigation3000 rcf, 00:02:00 (keep eluate as ‘flow through’ fraction).

2m
Place the column into a new tube, and load Amount300 µL of water, and centrifuge at Centrifigation3000 rcf, 00:02:00 (keep eluate as ‘wash’ fraction).

2m
Place the column into a new tube, and load the TMT wash solution (5% ACN, 0.1% triethylamine (TEA)) .
Place the column into a new tube, and load Amount300 µL of the appropriate elution solution (see table below), and centrifuge at Centrifigation3000 rcf, 00:02:00 to collect the fraction.

2m
Repeat step 5 for each step of the gradient fraction.
If you are concatenating the fractions, combine the fractions into the desired combinations

Lyophilise all samples until there are only a few µL left in the tube